too much salmon sperm? - what will happen? (Oct/11/2009 )

Hello,
I've just realised that I added 10X more salmon sperm to my ChIP than I was supposed too... ... i just can't brain today.

Any idea on what will happen?
Any ideas on how i can fix this?

V

vetticus3 on Oct 11 2009, 07:27 PM said:

In our plate based assay, increasing the salon sperm DNA beyond a certain point increased background. I don't know if it would do the same if you're using beads. In any case, there's not much to be lost by going through and completing the assay.

i pushed ahead anyway... but i've got my cells up and growing as an insurance plan. PCR in a few hours... we'll see. somedays, i swear, i don't see the decimal point.

hypothetically speaking, is it possible to "over" reverse cross link? say, if i left it for 18 hours ... i don't want to spend 14 hours days at work.

V

vetticus3 on Oct 12 2009, 11:59 PM said:

You can definitely overcrosslink. It can make shearing impossible if you crosslink long enough and it can mask epitopes as well (especially if they depend on lysine).

KPDE on Oct 15 2009, 08:25 PM said:

I thought he said over "reverse" crosslink

and regarding that, I think reverse cross link time can be quite flexible, and usually put at 65C with 10ul 5M NaCl overnight

haha, i have found my answer...
too much salmon sperm makes a bit more background, but nothing to get fussed over.

i didn't try to over reverse crosslink... i wussed out and waited for the 6 hours.

V